Liquid chromatographic/tandem mass spectrometric method for the determination of the tobacco-specific nitrosamine metabolite NNAL in smokers' urine

J Mass Spectrom. 2003 Jan;38(1):98-107. doi: 10.1002/jms.406.


A specific and rapid liquid chromatographic/tandem mass spectrometric (LC/MS/MS) method was developed and validated for NNAL, a metabolite of the tobacco-specific nitrosamine metabolite NNK. The metabolite was detected in smokers' urine with a limit of quantitation (LOQ) of 20 pg ml(-1) and a linear range up to 1000 pg ml(-1). The method features a single solid-phase extraction step and MS/MS monitoring following electrospray ionization. Fragmentation pathways for the protonated molecular ion are proposed. The sample preparation is simpler than that for gas chromatographic methods reported in the literature and maintains sensitivity adequate for determining NNAL in smokers' urine. By using enzyme hydrolysis to determine total NNAL in urine, the amount of NNAL-glucuronide was calculated. A standard pooled smokers' urine sample used for development gave values of 176 +/- 8 pg ml(-1) free NNAL and 675 +/- 26 pg ml(-1) total NNAL following enzyme hydrolysis. The method was applied to a group of seven smokers; the free NNAL level for the group was 101-256 pg ml(-1) with NNAL-glucuronides at 247-566 pg ml(-1). The ratio of conjugated to free NNAL was in the range 0.98-2.95. The variability in total daily amount of NNAL excreted (ng per 24 h) had RSDs of 6-21% for free NNAL, 7-22% for conjugated NNAL and 6-20% for total NNAL excreted. When normalized to the number of cigarettes smoked, the amounts of NNAL excreted per cigarette smoked were in the range of amounts of NNK yields reported for cigarettes in the literature.

MeSH terms

  • Adult
  • Biotransformation
  • Chromatography, High Pressure Liquid
  • Female
  • Humans
  • Male
  • Mass Spectrometry / methods*
  • Molecular Structure
  • Nitrosamines / isolation & purification
  • Nitrosamines / metabolism*
  • Nitrosamines / urine*
  • Sensitivity and Specificity
  • Smoking / urine*
  • Solutions / chemistry
  • Tobacco / chemistry
  • Tobacco / metabolism*


  • Nitrosamines
  • Solutions